Increased flux of free fatty acids (FFA) from adipose tissue to non-adipose tissues contributes to and augments many metabolic derangements that are characteristic of the metabolic syndrome and type 2 diabetes, including insulin resistance. Evidence indicates that adipocyte mitochondria may play an important role in adipocyte FFA sequestration, suggesting that abnormalities of these organelles may contribute to an increased FFA flux from adipose tissue. Recent animal studies and very limited human studies have demonstrated various abnormalities of adipocyte mitochondria in insulin resistance and reversal of most of these abnormalities by treatment with thiazolidinediones (TZD), but FFA flux has not been determined. Therefore, the overall goal of this project is to examine whether abnormalities of adipocyte mitochondria are associated with increased FFA flux in humans, and hence contribute to its related metabolic derangements. We will address the following Specific Aims: 1) To examine mitochondrial mass and density of isolated adipocytes in subjects that are resistant to suppression of FFA release by insulin and subjects that are sensitive to the suppression of FFA release by insulin. We will test the hypothesis that adipocyte mitochondrial mass or density is reduced in individuals with resistance of systemic FFA release to suppression by insulin. 2) To use HPLC-ESI-MS/MS proteomics to determine the relative abundance of mitochondrial proteins in adipocytes from subjects that are resistant to suppression of FFA release by insulin and subjects that are sensitive to the suppression of FFA release by insulin. We will test the hypothesis that there are differences in adipocyte mitochondrial protein abundance in individuals with resistance of systemic FFA release to suppression by insulin regardless of mitochondrial mass/density. 3) To use polarographic analyses to determine, in the presence of different substrates, the respiratory capacity and the sensitivity of respiratory control to energy demand of isolated adipocyte mitochondria from subjects that are resistant to suppression of FFA release by insulin and subjects that are sensitive to the suppression of FFA release by insulin. We will test the hypothesis that there are functional abnormalities of adipocyte mitochondria in individuals with resistance of systemic FFA release to suppression by insulin that correspond to alterations in protein abundance. Better understanding of the mechanisms of increased flux of FFA from adipose tissue is critical for developing treatment and prevention strategies for conditions related to increased plasma FFA levels, including type 2 diabetes. PUBLIC HEALTH RELEVANCE: Insulin resistance in skeletal muscle and liver plays a major role in the pathogenesis of impaired glucose tolerance and type 2 diabetes. Better understanding of the molecular mechanisms is critical for the development of therapeutic strategies and prevention.